This invention relates generally to novel methods for diagnosing and overcoming resistance to the macrocyclic lactone compounds. More specifically, the invention pertains to unique methods for detecting the development of resistance to macrocyclic lactones using nucleic acid probes and enhancing the efficacy of the macrocyclic lactones using multidrug resistant reversing agents.
Macrocyclic lactone compounds such as the LL-F28249 compounds, the milbemycins and the avermectins are widely used for treatment of nematode and arthropod parasites. The highly active LL-F28249 family of compounds are natural endectocidal agents isolated from the fermentation broth of Streptomyces cyaneogriseus subsp. noncyanogenus. U.S. Pat. No. 5,106,994 and its continuation U.S. Pat. No. 5,169,956 describe the preparation of the major and minor components, LL-F28249xcex1-xcex. The LL-F28249 family of compounds further includes, but is not: limited to, the semisynthetic 23-oxo derivatives and 23-imino derivatives of LL-F28249xcex1-xcex which are shown in U.S. Pat. No. 4,916,154. Moxidectin, chemically known as 23-(O-methyloxime)-LL-F28249xcex1, is a particularly potent 23-imino derivative. Other examples of LL-F28249 derivatives include, but are not limited to, 23-(O-methyloxime)-5-(phenoxyacetoxy)-LL-F28249xcex1, 23-(semicarbazone)-LL-F28249xcex1 and 23-(thiosemicar-bazone)-LL-F28249xcex1.
The milbemycins, also known as the B-41 series of antibiotics, are naturally occurring macrocyclic lactones isolated from the microorganism, Streptomyces hygroscopicus subsp. aureolacrimosus. U.S. Pat. No. 3,950,360 shows the preparation of the macrolide antibiotics milbemycinxcex11-xcex110, milbemycinxcex21-xcex23 etc. These compounds are also commonly referred to as milbemycin A, milbemycin B, milbemycin D and the like, or antibiotic B-41A1, antibiotic B-41A3, etc.
The avermectins, also known as the C-076 family of compounds, are naturally occurring macrocyclic lactones produced by the soil actinomycete microorganism, Streptomyces avermitilis. U.S. Pat. No. 4,310,519 discloses the isolation and preparation of the major components A1a (e.g., avermectin A1a), A2a, B1a and B2a, and the minor components A1b (e.g., avermectin A1b), A2b, B1b and B2b. The C-076 family additionally embraces the semisynthetic derivatives such as the 22,23-dihydroavermectins described in U.S. Pat. No. 4,199,569. The semisynthetic derivatives include, but are not limited to, ivermectin, abamectin, doramectin, eprinomectin and the like.
Resistance to all of the broad spectrum macrocyclic lactone compounds has been encountered in most regions of the world where the compounds are used routinely in animal production. For instance, drug resistance to ivermectin (IVM), chemically known as 22,23-dihydroavermectin B1 or 22,23-dihydro C-076 B1 and a commonly used member of the avermectin drug family, has become a widespread problem, particularly in nematodes of sheep, goats and cattle (Shoop, Parasitol. Today 9: 154-159, 1993). In some parts of the world, the survival of commercial animal production is threatened by the development of anthelmintic resistance. Additionally, there is conflicting evidence as to whether ivermectin (avermectin) resistance confers resistance to the related milbemycins or other macrolides (Arena et al., J. Parasitol. 81: 286-294, 1995; Oosthuizen and Erasmus, J. So. African Vet. Assoc. 64: 9-12, 1993; Pomroy and Whelan, Vet. Rec. 132: 416, 1993; Shoop, 1993; Condora et al., Vet. Rec. 132: 651-652, 1993; Pomroy et al., N.Z. Vet. J. 40: 76, 1992; Pankavich et al., Vet. Rec. 130: 241-242, 1992; Craig et al., Vet. Parasitol. 41: 329-333, 1992). The mechanisms of resistance to the avermectins, the milbemycins and other macrocyclic lactone compounds remain unknown.
P-glycoproteins (Pgp) were identified some years ago as proteins involved in multidrug resistance (MDR) of mammalian tumor cells (Julino and Ling, 1976; Gros and Buschman, 1993; Gotteesman and Pastan, 1993). MDR proteins may also be involved in drug resistance in the protozoal parasites Entamoeba histolytica (Whirth, Archivos De Investigacion Medica 21(Supp. 1): 183-189, 1990; Samuelson et al., Mol. Biochem. Parasitol. 38; 281-290, 1990), Leishmania enriietti (Chow, Mol. Biochem. Parasitol. 60: 195-208, 1993), L. dononani (Callahan et al., Mol. Biochem. Parasitol. 68: 145-149, 1994); and Plasmodium falciparum (Volkman et al., Mol. Biochem. Parasitol. 57: 203-211, 1993; Cowman et al., J. Cell Biol. 113: 1033-1042, 1991). While many researchers believe that the proposed mechanism for Pgp involvement in drug resistance is that Pgp behaves as a pump to increase drug efflux, Callahan et al. (1994) suggested that Pgp may work by decreasing drug influx. However, the whole picture of how Pgp can be responsible for drug resistance is still unclear.
Only recently have Pgp homologs been investigated in nematodes (Sangster, Parasitol. Today 10: 319-322, 1994; Lincke et al., EMBO J. 12: 1615-1620, 1993; Lincke et al., J. Mol. Biol. 228: 701-711, 1992). Three full length Pgp genes and one partial Pgp gene from the free-living nematode, Caenorhabditis elegans have been cloned, sequenced and mapped to chromosomes I, IV and X (Lincke et al., 1992). Sangster et al., J. Cell Biochem. 17 (Supp.): 1223, 1993, indicated evidence for several partial genes for Pgp in the parasitic nematode Haemonchus contortus, although sequence information was missing. In vivo experiments have shown that disruption of the mouse mdr1, a P-glycoprotein gene, leads to an impairment in the blood-brain barrier and to increased sensitivity to drugs in these mice (Schinkel et al., Cell77: 491-502, 1994). Mice with deletion of mdr1a were 50-100 times more sensitive to ivermectin than normal mice.
Drug resistance based on overexpression of P-glycoprotein has been shown to be reversed by verapamil and a number of other calcium channel blockers, calmodulin antagonists, steroids and hormonal analogs, cyclosporins, dipyridamole and other MDR-reversing agents (Ford, Hematol. Oncol. Clin. North Am. 9: 337-361, 1995). However, there has been no report or suggestion in the literature to use MDR-reversing agents to combat resistance in nematodes and arthropods to pesticides.
There is a definite need to understand the mechanism of macrocyclic lactone resistance, to be able to detect insipient resistance before it becomes flagrant and is difficult to manage the health of the animals. The ability to reverse the resistance has great potential for maintaining parasite control in the face of a failure of conventional treatment. An important object of the present invention, thus, is to determine these mechanisms of resistance in order to find viable, sensitive means to detect and to overcome the problematic resistance thereby improving parasite control.
Heretofore unknown, it is now found that the mechanism of resistance to the macrocyclic lactone compounds is due to overexpression of novel P-glycoprotein homologs. It is further newly found that the nucleic acid molecules encoding the P-glycoprotein homologs or the fragments thereof regulating this resistance are useful as unique probes in methods for diagnosing the resistance to the macrocyclic lactones. For the first time, the reversal of resistance to the macrocyclic lactone compounds using multidrug resistance reversing agents is described herein.